Annals of Phytomedicine 4(1): 94-98, 2015 ANNALS OF PHYTOMEDICINE 94 Journal homepage: www.ukaazpublications.com An International Journal

ISSN : 2393-9885

Evaluation of phytochemical investigation and immunomodulatory activity of four different species of vidari by carbon clearance test on wister rats V.K. Shilpashree, Raman Dang* and Kuntal Das* Karavali College of Pharmacy, NH-13, Opp: Mangalajyoti, Vamanjoor, Mangalore-575028, Karnataka, India *Department of Pharmacognosy and Phytochemistry, Krupanidhi College of Pharmacy, #12/1, Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bengaluru-560035, Karnataka, India

Received April 20, 2015: Revised May 10, 2015: Accepted May 15, 2015: Published online June 30, 2015

Abstract The present study was undertaken to evaluate immunomodulatory activity of vidari. Vidari is the name of four botanical sources of plant drug which is controversial in Indian market. They are namely; tuberose DC (P.t) (Family: Leguminosae), Jacq (I.m) (Family: Convolvulaceae), hondala de Wide (A.h) (Family: ) and Linn (C.c) (Family: Cycadaceae). In this study, preliminary investigations on phytoconstituents were evaluated separately and revealed the presence of carbohydrate, phenols, alkaloids, glycosides, saponins, phytosterol, flavonoids, wax and gums in all the examined drugs but tannins were absent in all the drugs. Furthermore, immune response was evaluated by carbon clearance assay (Granulopectic index) against standard marketed drug, Withania somnifera (W.s), an approved potent immunomodulatory agent (positive control). Wister rats of either sex were divided into 6 groups (6 animal each) and all the drugs were administered at dose of 250 mg/kg body wt. after calculated individual maximum tolerance dose as per OECD guidelines. All the test substances and standard showed significant activity compared to control (p < 0.001) and, thereafter, immune response with respect to granulopectic index of P.t (p > 0.05) was found better (0.051/ min), compared to other extracts but the value was lesser than that of positive control (0.053/ min). This study ascertained pharmacologically, the authenticity of four different species of plant belong to the same name of “vidari”.

Key words: Carbon clearance assay, immunomodulatory, phytochemicals, vidari, Withania somnifera

1. Introduction of herbal medicines. Dried plant products sold in the market are generally difficult to identify and at the same time, identification of A vast number of research investigations have described the use of first step in quality control of herbal medicines. Different herbal in traditional ayurvedic medicine for many years and have raw drugs are sold under the same name and create controversies recently gained tremendous focus in the field of pharmacological with respect to the botanical identities of drugs, available in the industries. The quality assessments of the biological properties of market. Among the plants known for medicinal value, the ayurvedic extracts from various plants can serve as a source of newer drug plants with the name of vidari are very important for their molecules which help in many areas of health related problems therapeutic potentials. Vidari has four different plant species with (Acuna et al., 2009) and several marketed plant products have been their respective botanical identities and family (Venkatasubramanian used for the treatment of different diseases (Figueroa et al., 2015). et al., 2009), namely; Pueraria tuberose DC (P.t) (Family: ), Many herbs enlisted as “Rasayana” drugs in Ayurveda are believed Ipomoea mauritiana Jacq (I.m) (Family: Convolvulaceae), Adenia to improve defense mechanisms of the body, promote physical and hondala de Wide (Family: Passifloraceae) and Cycas circinalis Linn mental health and enhance longevity (Shukla et al., 2009; Jantan et (C.c) (Family: Cycadaceae). The substitutes may or may not al., 2011; Kumar et al., 2012; Zhuang et al., 2012), among them resemble vidari in terms of morphology, properties or actions. Vidari Ocimum sanctum, W.s is well established as potent immune is an ingredient of Chyavanaprash, one of the top-selling products response plant drug (Caroline Jeba et al., 2011). In order to meet of ayurvedic industry (Venkatasubramanian et al., 2009). As per the rising demand for the raw drugs, adulteration and substitution the Ayurvedic Pharmacopoeia of India correlates vidari to P.t which have become frequent which in turn results in compromised quality is commonly known as “Vidarikand”. The plant is available throughout the India and it is traditionally used for bleeding disorders, decreased seminal quantity, purification of blood, tuberculosis, Author for correspondence: Dr. Kuntal Das Associate Professor, Krupanidhi College of Pharmacy, #12/1, Chikka cough, pain, burning micturition, herpes, rejuvenator, tonic, Bellandur, Carmelaram Post, Varthur Hobli, Bengaluru-560035, restorative, aphrodisiac, galactagogue, diuretic, demulcent, Karnataka, India haemorrhage, bronchial asthma and urinary disorders E-mail: [email protected] (Yoganarasimhan, 1996; The Ayurvedic Pharmacopoeia of India, Tel.: +91-9632542846 2006; Maji et al., 2014). But as per the local names of vidari, it Copyright @ 2015 Ukaaz Publications. All rights reserved. Email: [email protected]; Website: www.ukaazpublications.com 95 contains another three plants and they are used for different Mice (fasted for 3-4 h.) were divided into four groups, of two purposes like, tubers of I.m, A.h are used against sexual debility, animals (male and female) each for administration of each crude pain, inflammations, burning sensations and skin problems (Puri, drug. Mice were administered with test substances orally at the 2003) and C.c is used for high blood pressure, headaches, congestion, dose of 1g/kg body weight. Then the animals were observed for rheumatism, bone pain and treatment of estrogen-dependent tumors 24 h. for sign and symptoms of acute toxicity, viz. mortality, (Maria et al., 1995; Kalpashree and Raveesha, 2013). These activities tremors, alertness, eye movements and motility. Further, second are mainly due to the presence of several phytochemicals present dose of 2 g/kg body weight was given to the animals and observed in these plants, viz. puerarin, daidzein, genistein, genistein, for 72 h., which was also found to be safe and no toxic symptoms puerarone, coumarin, anthocyanin, lupinoside, tuberosin, were observed. W.s was used as standard at the dose of 250 mg/kg pterocarpintuberosin, puetuberosanol and hydroxytuberosone (Davis and Kuttan, 2000) and as per that recommended doses were (Maji et al., 2012; Sawale et al., 2013) among the phytochemicals, selected for all tested drugs at the dose of 250 mg/kg. the isoflavones puerarin, daidzein and genistein are most important for their immunomodulatory activity (Sawale et al., 2013) but no 2.6 Experimental protocol such concrete evidences are available for determination of the All the tested animals’, randomly divided into 6 groups of 6 rats pharmacological activity, especially comparative immunomo- each. dulatory activity for all four drugs. Based on the above context, the present study was designed to explore the immunomodulatory Group 1: Control animals potential of all four different plant drugs and to establish the potent Group 2: DC. (250 mg/kg) pharmacological authentication under the same local name of vidari. Group 3: Ipomoea mauritiana Jacq. (250 mg/kg) 2. Materials and Methods Group 4: de Wide (250 mg/kg) 2.1 Plant material Group 5: Cycas circinalis L. (250 mg/kg) Group 6:Withania somnifera (L.) Dunal standard treated animals Dried mature tubers of P.t, I.m, A.h and stems of C.c used for the (250 mg/kg) study were collected by qualified field botanists from different locations of Belgaum, Bengaluru local market (Karnataka), 2.7 Carbon clearance assay Wayanad, Kozhikode, Idukki (), Pune, Bheamashanka All the animals in the 2nd, 3rd, 4th, 5th and 6th group received 250 mg/ (Maharashtra) and Tirupati (Andhra Pradesh). The samples were kg suspension of drugs, P.t, I.m, A.h, C.c and W.s for a period of 5 authenticated by qualified plant taxonomists of Herbarium division consecutive days. After 48 hours of last dose of drug, blood sample of FRLHT. Voucher Specimen No. (P.t = L/07/02/032; I.m = L/07/ 02/035; A.h = L/07/10/027 and C.c = L/07/04/011) for all the samples was collected by retro-orbital puncture which was taken as blank were preserved at Raw Drug Collection Centre, FRLHT, Bangalore. (Tiwari et al., 2004; Zhao et al., 2005). Each rat received an intravenous injection of carbon ink suspension consisting of 3 ml 2.2 Preparation of extract of pelikan ink (Germany), 4 ml of saline and 4 ml of 3% gelatin Dried coarsely powdered tubers of P.t, I.m, A.h and stem of C.c solution (Morris et al., 2003) via the tail vein at a dose of 0.5 ml/ (250 g) were separately defatted with petroleum ether at 45oC for 100 gm body weight (Daswani and Yegnanarayan, 2002). Blood 5 h, using Soxhlet apparatus. The marc left was subsequently was collected immediately after i.v injection of carbon ink extracted with ethyl acetate, methanol and water (45-50oC) for 7 h. suspension and at an interval of 3, 6, 9, 12, and 15 min. 25 µl of The crude brown residue mass of extract was then concentrated by blood was added to 2 ml of 0.1% sodium carbonate solution to lyse using rotary flash evaporator and then stored at 4oC in refrigeration the erythrocytes. At the end of the blood collection, the absorbance condition in separate glass bottles. The percentage yield of extracts of each sample was measured at 660 nm, using spectrophotometer. were calculated on dry wet basis. The clearance value ‘K’ was calculated according to the following 2.3 Pharmacognostic studies equation: K= (log OD ) – (log OD ) / t – t All the above extracted samples were screened for various 1 2 2 1 phytochemical constituents’ presence as per the method described where ‘t1’ represents the time in minutes when the samples ‘OD1’ by Kokate (1997). were withdrawn. 2.4 Experimental animals The mean of the clearance values at different time intervals gives Wister rats of either sex were used in the present study. The animals the clearance value K (Granulopectic index) (min-1). were fed with standard pellet diet, water ad libitum and maintained 2.8 Statistical analysis under standard environment condition employed. They were o housed under standard conditions (22 ± 5 C with 12 h of light/dark Data were expressed as the mean ± standard deviation (S.D) and cycle). All experimental protocols were approved by Institutional statistical analysis was performed using ANOVA, followed by Animal Ethical Committee Clearance (AACP/IAEC/M-75/2006), Bornferronis multiple comparison test. Al-Ameen College of Pharmacy, Bangalore (Karnataka), India. 3. Results and Discussion 2.5 Maximum tolerance dose (MTD) 3.1 Yield of the extracts of vidari plants All the dried coarse powdered drugs were tested for MTD to determine the nature and extent of the untoward reactions which Percentage yield of all the extracted drugs were calculated and might follow the administration of a single dose (or overdose) of tabulated in the Figure 1. The results revealed that aqueous extract the drug, using swiss albino mice of either sex (25-30 g). of P.t yielded 3.2% w/w, followed by methanol extract (2.8% w/w) 96 whereas aqueous extract of I.m showed 2.6% w/w, followed by the cultural condition of the plant sources whereas several research 2.7% w/w for C.c. but the yield was less in A.h extracts than all references revealed that the yield of the plant drugs are varied with others. A.h aqueous extract showed 1.6% w/w, followed by 1.8 % the several conditions, viz. source, location, soil nature, parts use w/w yield in methanol extract. This result variation may be due to harvesting time, session and so on (Evans, 1996; Cavaliere, 2009; Namdeo et al., 2010).

Figure 1: Yiled of various extracts for all the different plant species of Vidari

P.t = Pueraria tuberose DC; I.m = Ipomoea mauritiana Jacq; A.h = Adenia hondala de Wide; C.c = Cycas circinalis Linn.; EA = Ethyl acetate; Met = Methanol; Aq = Aqueous

Table 1: Phytochemical study of various plants of vidari

P. t I. m A. h C. c C.C EA Met Aq EA Met Aq EA Met Aq EA Met Aq

Alk – + – – – + – – – – – –

Car – + + – + + + + + – + +

Gly – + + – + + – + + – + –

Sap – + + – – + – – + – – +

Ste – + + – + – – + – + + –

Fats – – – – – – – – – – – –

Res – – – – – – – – – – – –

Fla – + + – + + – + – – + +

Phe – + + – + – – – – – – –

Tan – – – – – – – – – – – –

Pro – – + – – + – – + – + +

Gum – + + – – + – – + – + +

EA = Ethyl acetate; Met = Methanol; Aq = Aqueous Alk= Alkaloids; Car = Carbohydrates; Gly= Glycosides; Sap= Saponins; Ste= Steroids; Res= Resins; Fla= Flavonoids; Phe= Phenols; Tan = Tannins; Pro= Proteins 97

Control Pueraria Ipomoea Cycas Adenia Withania tuberosa mauritiana circinalis hondala somnifera

Figure 2: Graphical representation of all test substances, standard and control on granulopectic index (min-1)

3.2 Screening of phytoconstituents and A.h also gave better activity as compared to control (0.034/ min) even though they are used as adulterants and substitutes in All the extracts for different plant species were screened for the the market. Similarities between phytoconstituents and bioactivity presence of the phytochemicals and the results reported that all the of P.t and I.m makes the latter as good substitute for the former. In plants exhibited some important secondary metabolites, viz. fact in Kerala, I.m known as Palmuttukku is used as vidari or carbohydrate, waxes, phenols, alkaloids, glycosides, saponins, kshiravidari (Venkatasubramanian et al., 2009). phytosterol, flavonoids and gums but absence of tannins. Furthermore, A.h and C.c showed absence of alkaloids and resins Bibliographic references revealed components such as (Table 1). The variation of these studies were may be due to the polysaccharides, lectins, proteins and peptides present in plants nature of plant sources which further correlated with the earlier have been shown to stimulate the immune system (Haijto et al, studies of Mamoon et al. (2013) and Kulkarni et al. (2013). 1989). Several immunomodulators like sterols, a group of compounds were identified from plants that have demonstrated 3.3 Maximum tolerance dose (MTD) promising results in a number of clinical trials. The most studied The MTD studies were carried out as per OECD guidelines on sterols are beta sitosterol and its glycosides (Patrick and Johan, swiss albino mice of either sex for all the powdered drugs. All the 1999). Furthermore, Zhao et al. (2005) have isolated and purified species used as vidari were tested at two different doses, viz. 1g/kg polysaccharide from Ipomoea batatas (Sweet potato) and and 2 g/kg bw, p.o. Toxic symptoms with the based on various characterized as (1  6)--D-glucan that showed in vivo immune parameters like mortality, tremors, alertness, eye movements and function of mouse. Administration of an extract from the powdered motility, convulsions and mortality were recorded after 24 and 72 root of the plant, W.s showed an enhancement in phagocytic activity h. and resulted that there are no changes on physiological behavior of peritoneal macrophages when compared to control in mice. These which was found to be safe dose and no toxic symptoms were results confirm the immunomodulatory activity of W.s extract, observed and resulted all the test substances were safe to use for which is a known immunomodulator in indigenous medicine (Davis various applications. and Kuttan, 2000). The results of the above surveyed literature confirms that the polysaccharide and sterol are might be the 3.4 Carbon clearance assay responsible for immunomodulatory activity. The test was conducted on wistar rats in 6 groups (n=6) to establish phagocytic activity of reticuloendothelial system after treatment 4. Conclusion with 250 mg/kg of the powdered mature tubers of P.t, I.m, A.h and Qualitative screening of phytochemicals for all four plant species stems of C.c as per the standard procedure where control group of vidari indicated the presence of carbohydrates, glycosides, received normal food and water and dried powder of W.s (250 mg/ saponins, phytosterols, flavonoids, proteins, phenols, gums and kg p.o) was used as positive standard and granulopectic index mucilages which are essential secondary metabolites for any (min-1) were calculated for all the test drugs. The results identified activities. Furthermore, alkaloids were found to be present in P.t that all the test substances and standard showed significant activity and I.m whereas resins, fats and tannins were absent in all the compared to control (p < 0.001). The activity of P.t (p > 0.05) was vidari species. These variations of the phytoconstituents resulted found to be very close (98.07%, 0.051/min) to that of positive variant immune response when compared to standard W.s powder standard of W.s, (0.053/min), followed by I.m (94.2%, 0.049/min) and revealed significant activity with P.t, followed by I.m due to whereas A.h showed least activity (80.76%, 0.041/min) compared similarities of their phytoconstituents but small variation of results to all other test substances (Figure 2). It was interesting that C.c due to the effect of cultural conditions. Hence, the present study 98 demonstrated the potential value of obtaining guidance from Maji, A. K.; Mahapatra, S. and Banerjee, D. (2014). In vivo immuno- traditional knowledge for development of quality standards through modulatory potential of standardized Pueraria tuberosa extract pharmacological activities for herbal medicines with the proper and its isoflavonoids. Int. J. Pharm. 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